InteractiveFly: GeneBrief

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

BIOLOGICAL OVERVIEW

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 sd^Gal4 control and small rough eyes when expressed under ey^Gal4 control. Expression of dan using EPg J3-220, under control of Dll^Gal4 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 Dll^Gal4 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 Dll^Gal4 driver. However when expressed using dpp^Gal4, 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. ptc^Gal4 directed expression of Dan causes strong suppression of the arista-to-tarsus transformation in the ss mutant antenna. ptc^Gal4 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 ptc^Gal4 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 dan^ems3 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 spineless^aristapedia. ss^a 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 ss^a function. The consequences of removing one copy each of Dan and Danr was examined in a ss mutant background. The spineless^114.4 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 danr^ex35 deletion in this background causes a modest increase in the size of the basal capsule and in the number of ectopic bristles. The dan danr^ex56 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 danr^ex56 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 danr^ex56 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 dan^ems3 allele lies at the same position as the arginine that is required for DNA binding by the Pogo pipsqueak motif. Thus, the dan^ems3 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 danr^ex35 single mutant phenocopies the dan danr^ex56 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 danr^ex35 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).

REGULATION

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 hth^c1 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 dpp^Gal4 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, dpp^Gal4 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 dpp^Gal4 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 spineless^aristapedia (ss^a) mutants. ss^a alleles appears to reduce ss activity specifically in the antenna. Dan expression was lost from the distal part of ss^a mutant antennal discs. Loss of Dan expression in ss^a 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 ptc^Gal4 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 CtBP^87De-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).

CtBP^87De-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 CtBP^87De-10/M mosaic individuals have more ommatidia than wild-type eyes. In contrast, whereas CtBP^87De-10,dan,danr^ex56 triple mutant clones are similar to CtBP^87De-10 single mutant clones in being larger than wild-type clones ahead of the furrow, CtBP^87De-10,dan,danr^ex56 adults have small rough eyes. This suggests either that the CtBP^87De-10,dan,danr^ex56 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).

DEVELOPMENTAL BIOLOGY

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, Dany^AAA still retains some function; it promotes spermatogenesis beyond the stage when dany null mutant spermatocytes arrest. Moreover, the intracellular localization of Dany^AAA 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).

REFERENCES

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

date revised: 21 November 2016

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