disconnected and disco-related: Biological Overview | References
Gene name - disconnected and disco-related
Cytological map position - 14B1-14B1 and 14B1-14B1
Function - zinc finger transcription factors
Symbol - disco and disco-r
Genetic map position - X:16,104,631..16,110,767 [-] and X:16,012,821..16,042,982 [-]
Classification - zinc finger transcription factors
Cellular location - nuclear
Though initially identified as necessary for neural migration, Disconnected and its partially redundant paralog, Disco-related, are required for proper head segment identitity during Drosophila embryogenesis. This study presents evidence that these genes are also required for proper ventral appendage development during development of the adult fly, where they specify medial to distal appendage development. Cells lacking the disco genes cannot contribute to the medial and distal portions of ventral appendages. Further, ectopic disco transforms dorsal appendages toward ventral fates; in wing discs, the medial and distal leg development pathways are activated. Interestingly, this appendage role is conserved in the red flour beetle, Tribolium (where legs develop during embryogenesis), yet in the beetle no evidence was found for a head segmentation role. The lack of an embryonic head specification role in Tribolium could be interpreted as a loss of the head segmentation function in Tribolium or gain of this function during evolution of flies. However, an alternative explanation is suggested. It is proposed that the disco genes always function as appendage factors, but their appendage nature is masked during Drosophila embryogenesis due to the reduction of limb fields in the maggot style Drosophila larva (Patel, 2007).
The Drosophila disco gene was initially identified as required for proper neural migration (Steller, 1987), and later work demonstrated that disco along with the paralog disco-r were required for embryonic pattern formation (Mahaffey, 2001; Robertson, 2004). Yet because mutations in disco affected migration of neurons during development of the adult and work by Lee (1991) indicated that disco was expressed in many of the imaginal discs, it was thought that the disco genes would have a role after embryogenesis. Furthermore, disco-lacZ from the enhancer trap line C50.1S1 has been used as a marker for leg joint formation. This study presents evidence that disco genes are conserved members of the insect proximal/distal appendage specification network. In both Drosophila and Tribolium, these genes are expressed in the ventral appendages. Loss-of-function evidence from Drosophila and Tribolium indicates that the disco genes are required for ventral appendage development. Clonal analyses indicated that cells homozygous for Df(1)ED7355 were lost from adult tissues that developed from disco-expressing regions of the ventral imaginal discs, though such cells were viable elsewhere. There is no evidence that any genes in Df(1)ED7355 other than disco and disco-r affect viability or pattern formation. The proteins encoded by these other genes are unlikely to have such effects, and none are known to be expressed in a pattern similar to discoand disco-r. Therefore, it would be quite unlikely that one of these other genes would have an effect only in disco expressing regions while being viable elsewhere. Furthermore, clonal analysis in Drosophila and RNAi experiments in Tribolium yielded complementary results. It is unlikely that two non-specific processes could yield such similar results. Gain-of-function studies in Drosophila demonstrate that ectopic disco transforms dorsal appendages to ventral fates. Altogether, this is strong evidence that the disco genes are ventral appendage factors. Though the C50.1S1 enhancer trap has been used by others to mark the region of leg joint formation, it was not possible to test a direct role for the disco genes during leg joint formation, since the clones of Df(1)ED7355 cells did not survive in the leg discs. However, no indication was found of additional leg joints forming due to ectopic expression. The high levels of β-gal from the disco-lacZ enhancer trap C50.1S1 present in the leg joint regions may reflect the perdurance of β-gal (Patel, 2007).
That cells lacking disco function either fail to proliferate or die when they are in the medial to distal portions of the appendage primordia implies that they are recognized as aberrant or inappropriately determined cells which are removed through autonomous actions or by their normal counterparts. Cell communication is important in establishing appendage regions, and observations from this study demonstrate that there must be some form of communication between disco-expressing and non-expressing cells in the developing medial appendage region. Identifying the mechanism responsible is an important quest for the future (Patel, 2007).
Though there was no attempt to extensively address the regulatory interactions between the Disco proteins and other appendage factors, some insights are apparent. Tsh represses disco and disco-r during Drosophila embryogenesis (Robertson, 2004). That disco transcripts were not expressed throughout all of the proximal leg discs may indicate that Tsh represses the disco genes during appendage morphogenesis as well. In addition, that ectopic disco induced dac expression in the wing discs might indicate that dac is a target of Disco, though other explanations are certainly possible. At this time it is not known whether these regulatory events are direct (Patel, 2007).
During Drosophila embryogenesis, the disco genes act in parallel with the hox genes to establish proper segment identity in the head (Robertson, 2004). In this role, the disco genes are similar to the teashirt gene (tsh), which during embryogenesis encodes a trunk segment specification factor. Both the disco genes and tsh encode regionally expressed zinc finger transcription factors and, strictly from studies of Drosophila embryogenesis, they appear to establish zones along the anterior posterior axis of the embryo (Disco, head; Tsh, trunk) (Robertson, 2004). Interaction between these two systems is evident in that tsh expression represses disco and disco-r, limiting their expression in the trunk segments (Patel, 2007).
The newly discovered appendage role for the disco genes is intriguing in light of the segment specification function during Drosophila embryogenesis. tsh also is required for proper proximal development of adult appendages. As was found with disco, the appendage role of tsh appears to be conserved, while the trunk segment specification role is found only during fly embryogenesis. It is possible that the embryonic and appendage functions are distinct. If so, this would indicate that, for disco, either the head specification role was newly acquired in the fly lineage, or that it was lost in the beetle. Yet even during embryogenesis, it was not found that the disco genes are required for the appendage primordium and for the Keilin's organs, which are proposed to be remnants of larval appendages. Therefore, even during Drosophila embryogenesis, disco is functioning as an appendage factor. But what about the expression in the head segments? To address this, the differences between Drosophila and other insects as well as between the larval and adult forms of the fly are considered in this study. One suggestion: perhaps disco is always an appendage factor, including during specification of the Drosophila larval head segments (Patel, 2007).
Most insects have well-formed appendages when they hatch as larva, but this is not the case for the worm-like larva of higher dipterans. In these insects visible appendages do not arise until the pupal stage when the adult body develops. Appendages arise from the imaginal discs, which are blocks of cells set aside during embryogenesis. Certainly, reduction of the distal and medial appendage domains could account for the Keilin’s organs being derived from larval legs. Less obvious, but perhaps more significant in terms of novelty, are the changes that occurred to generate the internalized larval feeding apparatus of fly larvae. In Drosophila larvae, the embryonic head segments are highly reduced and internalized, unlike most other insects. Perhaps, to form an internalized, multi-segmental feeding apparatus, the mouthpart appendages (mandibles, maxilla and labial palps) have been reduced, as occurred with the legs. However, instead of reducing the medial and distal portions of the appendage, perhaps in the head the proximal tissues were reduced so that the medial appendage domain, governed by disco, remains and is prominent in these head segments. In this regard, it is interesting to note that in less highly derived insects with external larval head appendages, homologs of tsh are expressed in the ventral portion of the head segments. If this model of the evolution of the Drosophila larval head is correct, then the disco genes would have analogous roles as appendage factors in the head and trunk segments, also uniting their roles in establishing appendages in the larva and adult (Patel, 2007).
The C2H2 zinc-finger-containing transcription factors encoded by the disconnected (disco) and teashirt (tsh) genes contribute to the regionalization of the Drosophila embryo by establishing fields in which specific Homeotic complex (Hom-C) proteins can function. In Drosophila embryos, disco and the paralogous disco-related (disco-r) are expressed throughout most of the epidermis of the head segments, but only in small patches in the trunk segments. Conversely, tsh is expressed extensively in the trunk segments, with little or no accumulation in the head segments. Little is known about the regulation of these genes; for example, what limits their expression to these domains? This study reports the regulatory effects of gap genes on the spatial expression of disco, disco-r, and tsh during Drosophila embryogenesis. The data shed new light on how mutations in giant (gt) affect patterning within the anterior gt domain, demonstrating homeotic function in this domain. However, the homeosis does not occur through altered expression of the Hom-C genes but through changes in the regulation of disco and tsh (Sanders, 2008).
disco and disco-r, referred to together as the disco genes, and teashirt (tsh) are differentially expressed in the embryonic head and trunk segments and are therefore markers for head and trunk segment types. In the head segments the disco genes are required for the proper development of the larval mouthpart structures (Mahaffey, 2001; Robertson, 2004), while in the trunk segments, these genes are necessary for development of the Keilin's organs, small thoracic sensory structures and some peripheral neurons (Robertson, 2004; Patel, 2007). By contrast, tsh is necessary for proper development of most of the ventral trunk epidermis. Both the disco genes and tsh are also members of the proximal-distal patterning network (see Patel, 2007). The disco and tsh genes encode C2H2 zinc-finger transcription factors that are expressed early in embryonic development with precise, nearly reciprocal expression patterns in the trunk and head segments, but not much is known as to how these patterns are established. What is known is that ectopically expressing tsh in the head segments converts the expression of the disco genes to a trunk-like pattern. The Spalt major (Salm) protein represses tsh expression in the posterior labial segment, but otherwise little is known regarding the regulation of disco and tsh—in particular, what factors distinguish the head and trunk modes of expression. The gap genes are logical candidates for this role (Sanders, 2008).
Patterning the Drosophila embryo involves initial establishment of the axes, regionalization of the embryo, definition of the segments and their polarity, and the specification of unique identities to each segment. The early acting components of this genetic cascade include both maternally and zygotically expressed genes that set in motion the segmentation and segment identity processes. The gap genes are among the earliest zygotic factors involved in these processes. Regulated by maternal morphogens in the blastoderm, and by one another, these genes act via overlapping gradients to divide the embryo into broad regions and to regulate the expression of downstream segmentation genes (Sanders, 2008).
Comparative studies in other insects have revealed significant conservation in the function of many segmentation genes, but less clear is the functional conservation of the gap genes between insect species. Earlier studies -- one in Tribolium castaneum, examining a giant (gt) homolog (Tc'giant) (Bucher, 2004), and one in Oncopeltus fasciatus, examining a hunchback (hb) homolog (Of'hb) (Liu, 2004) -- conclude that the function of these gap genes is one of segmentation and segment identity, differing somewhat from the segmentation function characterized in Drosophila. This difference is likely due to the differential patterning of long vs. short germ-band insect embryos. The results presented in this study indicate that Drosophila gt, in fact, has an embryonic segment identity role similar to that observed in Tc'giant. Surprisingly, this identity function arises not only through changes in homeotic (Hom-C) gene expression, but also from the regulation of disco and tsh. This, in conjunction with other gap genes, defines the position of the embryonic head and trunk segment types (Sanders, 2008).
To explore the regulation of the disco gene during embryogenesis, disco mRNA accumulation was studied in homozygous gap mutant embryos. disco is normally expressed in the clypeolabrum, the optic lobe region, the antennal segment, the gnathal segments (mandibular, maxillary, and labial), the embryonic leg primordia, and transiently in similar positions in the abdominal segments and the proctodeum. Five homozygous gap gene mutants exhibited altered disco mRNA distribution -- hunchback (hb), Krüppel (Kr), giant (gt), tailless (tll), and caudal (cad). Of these, hb, Kr, and gt affected the gnathal/thoracic disco expression domains. disco-r mRNA accumulation was examined in hb12, Kr2, gtQ292, and gtX11 mutant embryos. Alterations in disco-r expression mirrored those of disco. Because the effects on disco and disco-r mRNA accumulation appeared to be identical, the remaining studies focused on the regulation of disco. It is noted that gt mutations had a particularly interesting effect, indicating a central role for gt in disco regulation and possibly head-trunk boundary formation. Therefore, this study concentrated on gt. Indeed, the effects of hb and Kr could be interpreted through their known cross-regulation of gt (Sanders, 2008).
Two significant conclusions are drawn from this study: (1) In its anterior expression domain, gt acts in both segment identity and segmentation roles, and these two roles are functionally separable; and (2) the distinction between the gnathal and trunk segment types is determined by the gap genes and is reflected by the head and trunk expression patterns of disco and tsh, which appear to be regulated by a series of repressive interactions (Sanders, 2008).
The assertion that gt acts in both segment identity and segmentation is based upon the following observations:
Initial characterizations of the gt mutant phenotype, based on SEM studies, described a fusion between the labial, first thoracic, and second thoracic segment, which, later in development, resolved such that the first and second thoracic segments separated, but the labial segment remained fused with the thoracic segment. Petschek (1990) describes the loss of the third (labial) En stripe as indicating the deletion of the labial posterior compartment, and it was suggested that this may be the extent of the 'gap' phenotype in the anterior gt domain. The current examination of gtQ292 embryos revealed clear indications of a homeotic transformation of the labial segment to a first thoracic identity. Mohler (1989) noted the presence of ectopic hairs in the dorsal cuticle of gtX11 mutants, but did not relate this observation to a change in segment identity (Sanders, 2008).
Larsen (2003) determined that En-expressing cells were the first to regress during the formation of the segment groove. He describe the absolute requirement for En expression in cells adjacent to the developing groove. Thus, in gt mutant embryos, the amount of En accumulation retained in the posterior labial compartment likely determines the extent of segmentation that will occur between the labial and first thoracic segments. Embryos hemizygous for gtX11 almost completely lose the third En stripe, coinciding with the virtually complete fusion between the labial and first thoracic segments in these individuals. Consequently, a duplication of the first thoracic segment was never observed in embryos of this genotype. This lack of ventral denticle duplication in gtX11 embryos follows when considering the loss of En in the posterior labial segment. Since more of the labial En stripe remains in gtQ292 embryos, a segment border can form. Interestingly, both gtX11 and gtQ292 develop ectopic dorsal hairs anterior to the first thoracic segment. En staining revealed that at least a portion of the dorsal ridge fuses (or never properly separates) from the dorsal labial segment, creating a segment that resembles the first thoracic segment. It is likely that the ectopic dorsal hairs arise from the dorsal ridge, which has been transformed toward dorsal first thoracic identity (Sanders, 2008).
The case for a gt segment identity function is strengthened by the alterations in the homeotic genes expressed in the gnathal and thoracic regions. In all gtQ292mutants examined, the labial segment expressed Scr, Antp, and tsh. This combination of segment identity factors is normally found in the first thoracic segment. Further, the labial segment shows significant reduction or alteration in pb and disco expression, both markers of gnathal identity (Sanders, 2008).
There are two potential explanations for the differential effects on En accumulation and the ventral cuticle phenotype in the gt alleles that were examined. First, the available gtQ292 stock may, over time, have acquired second site suppressors responsible for the occasional persistence of En accumulation in the posterior labial segment. However, when this allele was crossed into a different genetic background, the presence of En accumulation and ventral cuticle transformation was still observed. If it is a second site suppressor of the gt mutation, then it must lie on the X chromosome carrying the gtQ292 allele. A second possibility is that the gtQ292 allele is a strong hypomorph, rather than an amorphic allele, and the residual Gt function is sufficient in some individuals to allow the labial En segmentation process to proceed, although the segment identity process remains faulty. Regardless of which explanation proves to be true, it appears that the anterior gt domain regulates embryonic patterning at two different levels -- segmentation and segment identity -- and that these two processes are functionally separable from one another (Sanders, 2008).
This conclusion is not without precedent. Early reports suggested a possible homeotic function in addition to the segmentation function of Gt in its posterior expression domain (Harding, 1988; Mohler, 1989). Homeotic transformations and segmentation defects are observed in the mutant phenotypes of other gap genes. For example, in hb mutants, the loss of mid-abdominal segmentation is accompanied by mirror image duplications. The current results are significant, as they are the first to definitively demonstrate a segment identity role of the anterior gt domain (Sanders, 2008).
A recent study characterized the expression and function of a gt homolog in Tribolium (Tc'gt) (Bucher, 2004). As in Drosophila, Tc'gt mRNA is expressed in two primary domains -- one in the anterior of the embryo, overlying the gnathal region, and a second in the region of the third thoracic segment to the second abdominal segment. Although the anterior Tc'gt domain is similarly placed as compared to Drosophila, the posterior domain is shifted forward approximately five segments. RNA interference and morpholinos were used to knock down the expression of Tc'gt to explore its function. Interestingly, it was found that Tc'gt has a role in the identity specification of the maxillary and labial segments, but did not have a role in segmentation. The maxillary and labial segments were transformed to a first and second thoracic identity, respectively, while all three thoracic segments exhibit a third thoracic identity. There was no loss of the gnathal Tc'engrailed (Tc'En) stripes, although thoracic and abdominal Tc'En accumulation was affected to varying degrees in different embryos. Although the region affected by the loss of Tc'gt function is broader than the transformed region observed in Drosophila gtQ292 mutants, the nature of the homeotic change is quite similar. A gnathal segment(s) is transformed to a thoracic identity, and this identity change is separate from the segmentation process. The segment identity function for gt may have been present in the last common ancestor of the holometabolous insects, and the segmentation role of the anterior gt might have been acquired separately to accommodate the long germ-band mode of development (Sanders, 2008).
In the head, disco is expressed in most cells of the segmental epidermis, while there is little or no expression of tsh. By contrast, tsh is expressed throughout most of the trunk segmental epidermis, while disco is limited to the limb primordia. The genetic studies presented in this study demonstrate that the difference between head and trunk expression patterns, and therefore segment types, is dependent upon the gap genes, and particularly, on gt (Sanders, 2008).
In gt mutant embryos, both disco and tsh expression are altered reciprocally. disco expression is severely reduced in the labial segment and in fact is altered such that the remaining disco mRNA resembled the embryonic limb primordia expression observed in the thoracic segments. There is a concomitant expansion of tsh expression into the labial segment. It has been demonstrated that UAS-driven ectopic tsh expression in the gnathal segments reduces and alters disco expression such that it mimics the expression pattern of the thoracic segments (Robertson, 2004). Similarly, in gt mutants, it is the expansion of tsh expression into the labial segment that is responsible for the changes in disco expression. When both gt and tsh are absent, disco expression in the labial lobe recovers significantly, and the overall morphology of the labial segment and adjoining dorsal ridge is notably improved (Sanders, 2008).
The results may support a direct role for gt in the regulation of tsh. Although previous work demonstrated the requirement of Antp for appropriate tsh expression in the thoracic segments, and the loss of gt results in ectopic Antp protein in the labial segment, Antp is not required for ectopic tsh activation in the labial and maxillary segments of gt mutants. It is likely that gt directly limits the anterior expression of both tsh and Antp. Gt functions as a short-range repressor and has been shown to bind with high affinity to the CD1 sequence (TAT GAC GCA AGA) derived from the Kr regulatory region. There is a sequence ~0.5 kb upstream of the transcription start site of tsh that is similar to the CD1 sequence (TAT GAA GGA AGG), differing by only three bases. Although it remains to be investigated as to whether the Gt protein can bind to this sequence, the similarity in sequence to a known in vivo Gt-binding site supports direct repression of tsh by Gt (Sanders, 2008).
The results outline a model for the positioning of the gnathal/trunk boundary in the Drosophila embryo, involving a network of repressive factors. gt is a key player in this model. The anterior domain of gt is limited by its interactions with the zygotic gap genes hb and Kr, both of which act as repressors of gt expression. Gt in turn limits tsh expression, preventing expression in the labial segment. tsh expression is further limited by the expression of salm in the anlagen of the maxillary and labial segments. However, the results demonstrate that salm alone is insufficient for repressing tsh in the posterior labial segment in embryos lacking gt function. In the trunk segments, Tsh limits disco expression to only the embryonic appendage primordial, so that, lacking Tsh, disco expression is expanded through much of the gnathal segments (Sanders, 2008).
Questions remain regarding the activation of tsh and disco. disco mRNA accumulates in the cellular blastoderm prior to gastrulation, implying the involvement of maternal factors or early acting gap genes. However, none of the gap mutants that were tested affected the initiation of the initial anterior disco domain. disco was significantly affected by the loss of maternal bcd. This suggests that bcd and/or maternal hb may play a role in the initial activation of the anterior disco domain, after which Tsh acts to limit the disco to the gnathal region. tsh expression initiates prior to gastrulation, first with a central stripe that resolves to form a striped pattern reminiscent of the pair-rule genes. Again, none of the gap genes that were examined eliminated tsh expression. Although Kr is expressed in the central region of the embryo, where tsh is first transcribed, it is not the activator of tsh. Early tsh may respond to maternal factors and/or a combination of gap gene products in a concentration-specific manner, which would account for the inability to detect a single activator in gap mutant studies. Finally, although several instances were found where Tsh represses disco, there is no evidence that the reverse is true. What leads to the repression of tsh and concomitant maintenance of disco in the maxillary segment of gt mutant embryos is unclear at this time (Sanders, 2008).
During animal development, the HOM-C/HOX proteins direct axial patterning by regulating region-specific expression of downstream target genes. Though much is known about these pathways, significant questions remain regarding the mechanisms of specific target gene recognition and regulation, and the role of co-factors. From studies of the gnathal and trunk-specification proteins Disconnected (Disco) and Teashirt (Tsh), respectively, evidence is presented for a network of zinc-finger transcription factors that regionalize the Drosophila embryo. Not only do these proteins establish specific regions within the embryo, but their distribution also establishes where specific HOM-C proteins can function. In this manner, these factors function in parallel to the HOM-C proteins during axial specification. In tsh mutants, disco is expressed in the trunk segments, probably explaining the partial trunk to head transformation reported in these mutants, but more importantly demonstrating interactions between members of this regionalization network. It is concluded that a combination of regionalizing factors, in concert with the HOM-C proteins, promotes the specification of individual segment identity (Robertson, 2004).
disco was initially identified in a screen for mutations affecting neural development. It was not until the discovery of disco-related (disco-r) that a patterning role was uncovered (Mahaffey, 2001). The phenotype of terminal embryos lacking disco and disco-r is similar to those lacking the gnathal HOM-C genes Dfd and Scr; that is, structures from the gnathal segments (mandibular, maxillary and labial) are missing. This phenotype is due to reduced expression of Dfd and Scr target genes. Since HOM-C protein distribution is normal in disco, disco-r null embryos, and vice versa, these factors appear to act in parallel pathways (Robertson, 2004).
These studies have been extended and it is shown that: (1) Dfd can only direct maxillary developmental when Disco and/or Disco-R are present; (2) Tsh represses disco (and disco-r), helping to distinguish between trunk and gnathal segment types, and thereby establishing domains for appropriate HOM-C protein function, and (3) when ectopically expressed in the trunk, Disco represses trunk development and may transform these segments towards a gnathal segment type (Robertson, 2004).
Though HOM-C genes have a clear role in establishing segment identities, ectopic expression often has only a limited effect. The data indicate that, for Dfd, this restriction arises because of the limited distribution of Disco in the trunk segments. There are two important conclusions from these observations: (1) the spatial distribution of Disco establishes where cells can respond to Dfd, and this is probably true for Scr as well. Cells expressing disco develop a maxillary identity when provided with Dfd, even though this may not have been their original HOM-C-specified fate. This highlights (2) -- the combination of Disco and Dfd overrides normal trunk patterning, without altering expression of tsh and trunk HOM-C genes. As with the maxillary segment, identity is lost in the mandibular and labial segments when embryos lack disco and disco-r. This indicates that Disco and Disco-R may have similar roles in all gnathal segments. That co-expression of Disco and Scr in the trunk activates the Scr gnathal target gene pb strengthens this conclusion. Therefore, it is proposed that Disco defines the gnathal region, and establishes where the gnathal HOM-C proteins Dfd and Scr can function (Robertson, 2004).
Alone, ectopic Disco significantly alters development, indicating that Disco has a morphogenetic ability, separate from gnathal HOM-C input. Since Disco is required for normal gnathal development, it is suspected that disco specifies a general gnathal segment type. Definitive identification is difficult because of the lack of morphological or molecular markers that denote a general gnathal segment type. Yet, there is support for the conclusion that disco expression establishes a gnathal segment type. Ectopic Disco can, to some extent, override the trunk specification system and repress trunk development (repressing denticles, oenocytes and trachea). Furthermore, ectopic Disco blocks dorsal closure, which is similar to the role of endogenous Disco in the gnathal segments (Robertson, 2004).
Perhaps the most compelling evidence that Disco specifies a gnathal segment type comes from the observation that disco is activated in the trunk segments when embryos lack Tsh. The identity of the trunk segments in tsh mutant embryos is somewhat uncertain. It has been suggested that some aspects of the tsh phenotype indicate the trunk segments acquire gnathal characteristics; for example, the ventral neural clusters appear to be transformed to a gnathal-like identity. Mutations in the tsh gene can therefore be interpreted in two ways -- either they partially transform the trunk segments into a gnathal-like identity, and in particular the prothoracic segment into a labial one, or they cause a non-specific change in segmental identity perhaps due to cell death. However, the loss of tsh and the trunk HOM-C genes may transform the trunk cuticle toward anterior head cuticle. Again, the difficulty in assigning an identity is due to the lack of a readily discernable gnathal morphological or molecular marker. Evidence is presented that disco and disco-r are reliable molecular markers for gnathal identity, and disco mRNA is shown to be present in the ventral and lateral regions of the trunk segments in tsh mutant embryos. This expression of disco coincides, spatially, with the region of the trunk that is transformed in tsh mutant embryos. UAS-driven disco does mimic some aspects of tsh mutants, denticles are reduced and the ventral chordotonal neurons do not develop, but since Tsh is still present, the transformation caused by ectopic disco may be incomplete. Finally, Dfd cannot induce maxillary structures, even in tsh mutants, when disco and disco-r are absent. This reinforces the role for Disco in establishing gnathal identity, and indicates that the ectopic Disco present in embryos lacking Tsh is functional. Therefore, considering these arguments, it is proposed that Disco and Disco-R establish the gnathal region of the Drosophila embryo, and in this regard, they function similarly to Tsh, which specifies the trunk region (Robertson, 2004).
There are other parallels between Disco/Disco-R and Tsh. They are regionally expressed zinc-finger transcription factors, and they are required in parallel with the HOM-C proteins for proper segment identity. Furthermore, the distribution of these proteins establishes domains in which specific HOM-C proteins can properly direct embryonic development. The data reveal a regulatory relationship between Tsh and disco (and disco-r), indicating they are part of an interacting network that helps regionalize the Drosophila embryo. The HOM-C proteins then establish specific segmental identities in the appropriate region. In the trunk segments, Tsh, along with the trunk HOM-C proteins, specifies the trunk segment characteristics, in part by repressing disco and, thereby, preventing gnathal characteristics from arising in the trunk segments. The presented model requires that tsh expression be limited to the trunk segments, and it is proposed this is accomplished by another C2H2 zinc-finger protein, Salm. tsh expression has been shown to expand into the posterior gnathal and posterior abdominal segments in embryos lacking Salm. Therefore, Salm establishes the boundary between the Tsh and Disco domains. It is stressed that, at this time, it is not known what parts of this regulation are direct. Interestingly, other zinc-finger transcription factors are responsible for positioning salm expression, so that a more extensive hierarchy of zinc-finger transcription factors leads to regionalization, eventually establishing the domains of HOM-C protein function. It is also noted that Tsh has other roles than just repressing disco. Tsh actively establishes the trunk region, just as Disco does the gnathal. It is also noteworthy that ectopic Tsh activates disco in the labial sense organ primordia, leading to a Keilin's Organs fate, as occurs in the thoracic segments. Therefore, for unknown reasons, Tsh changes from a repressor of disco to an activator in these cells. This observation highlights the complex interplay between factors like Tsh and Disco, and it will be interesting to determine what causes these opposing roles (Robertson, 2004).
Many other questions remain. For example, how are the expression domains for these factors established? It is clear that Salm could form a boundary separating gnathal from trunk, but in salm mutants, tsh is only ectopically activated in the posterior labial segment, not in every gnathal segment. This implies that Salm forms a boundary, not by repressing tsh throughout the head, but by, in a sense, drawing a line between the head and trunk regions. What then prevents tsh expression from crossing that line and extending further into the gnathal segments in salm mutants? Is there an activator of tsh that is limiting, another gnathal repressor, or is something else involved? Likewise, what activates tsh and disco? It is unlikely that lack of Tsh is the only requirement for disco expression. More likely, this relies on the prior segmentation pathway. With regard to the HOM-C specification of segment identity, questions remain as to how the zinc-finger proteins establish where specific HOM-C proteins can function. Are the zinc-finger proteins co-factors or simply a parallel pathway? Furthermore, if they are co-factors for the HOM-C proteins, how can different HOM-C proteins establish different segment identities with the same co-factor (for example, Dfd and Scr with Disco), or how can different co-factors alter the role of a HOM-C protein (Scr with Disco or Tsh) (Robertson, 2004)?
Finally, the question remains of whether or not factors such as Disco and Tsh establish head/trunk domains and delimit HOM-C protein function only in the Drosophila embryo, in all stages of Drosophila or in other animals as well. Though this remains to be tested experimentally, there are indications that this may be a general mechanism. homologs of these zinc-finger genes are found in vertebrates and in other invertebrates, and, although only limited data are currently available, expression data indicate that these genes may have similar roles to their Drosophila counterparts during embryonic patterning. In an informative experiment the Tribolium Dfd homolog, Tc-Dfd, has been expressed in Drosophila embryos lacking the endogenous Dfd gene; persistent expression of Tc-Dfd rescues maxillary development. Though at present, it is not known whether or not a direct interaction is required between Disco and Dfd, this result would indicate that the Tribolium Dfd protein can fulfill the same roles as the Drosophila protein, and, therefore, it must be able to function with the Drosophila regionalization system. In any case, it will be important to investigate and interpret the role of the regionalizing genes as they relate to development and evolution of body pattern in other animals, and to ask whether a similar network is involved in patterning all animals (Robertson, 2004).
HOM-C/hox genes specify body pattern by encoding regionally expressed transcription factors that activate the appropriate target genes necessary for differentiation of each body region. The current model of target gene activation suggests that interactions with cofactors influence DNA-binding ability and target gene activation by the HOM-C/hox proteins. Currently, little is known about the specifics of this process because few target genes and fewer cofactors have been identified. A deficiency screen in Drosophila melanogaster was undertaken in an attempt to identify loci potentially encoding cofactors for the protein encoded by the HOM-C gene Deformed (Dfd). A region of the X chromosome was identified that, when absent, leads to loss of specific larval mouthpart structures producing a phenotype similar to that observed in Dfd mutants. The phenotype is correlated with reduced accumulation of mRNAs from Dfd target genes, though there appears to be no effect on Dfd protein accumulation. These defects are due to the loss of two functionally redundant, neighboring genes encoding zinc finger transcription factors, disconnected and disco-related. The role of these genes during differentiation of the gnathal segments is discussed and, in light of other recent findings, it is proposed that these regionally expressed zinc finger proteins may play a central role with the HOM-C proteins in establishing body pattern (Mahaffey, 2001).
Presence of either gene product is sufficient for normal development of the mandibular, maxillary, and labial lobes, but absence of both gene products disrupts development in these regions. The phenotype of terminal larvae lacking these two genes is strikingly similar to that of larvae lacking the HOM-C genes Dfd and Scr. disco was identified earlier as encoding a protein required for the formation of certain neural connections during embryonic and adult development of Drosophila. This does not appear to be a redundant function, because the phenotype was no more severe in Df(1)19 hemizygous embryos that lack both disco and disco-r. At present, it is not known whether disco-r also has an independent role (Mahaffey, 2001).
disco and disco-r encode proteins containing paired zinc finger domains, Disco with one pair while Disco-r has two pairs. The near identity of the Disco zinc finger pair and the first pair in Disco-r indicates that these proteins may bind to the same DNA sequence. This, along with overlapping distribution of mRNAs, likely explains the redundancy. However, the putative Disco-r protein contains a second pair of zinc fingers, and it is possible that these also influence DNA binding. If so, there may be some differences in the recognition site of these two proteins and, possibly, differences in their roles during development. It is worth noting that a mammalian gene, basonuclin, has been identified that encodes a protein with zinc finger domains similar to those in Disco; Basonuclin contains three pairs of zinc fingers, so in this respect it is more similar to the Disco-r protein. An ORF is also present in the Caenorhabditis elegans genome that encodes a peptide containing a single pair of zinc fingers quite similar to those in Disco; however, at this time little is known of the gene. Finding similar proteins in animals widely divergent from Drosophila indicates that at least some functions of Disco and/or Disco-r may be conserved during evolution (Mahaffey, 2001).
One may wonder whether disco and disco-r are head gap genes. The early distribution of disco mRNA may be suggestive, it is unlikely for the following reasons. Loss of disco and disco-r does not appear to cause a gap phenotype. No loss of segments is observed; the gnathal lobes form as expected. In addition, no change was observed in the distribution of the Engrailed protein in the gnathal cells until head involution is underway, and then the changes appear to be due to improper migration of the gnathal lobes in the mutant embryos. Further, disco-r function is sufficient for normal gnathal development, yet accumulation of disco-r mRNA in gnathal cells occurs well after segmentation. Finally, the process of segmentation in the gnathal region follows that of the trunk, relying on the gap, pair rule, and segment polarity functions. Taking this into consideration, it seems unlikely that disco and disco-r are head gap genes (Mahaffey, 2001).
However, it is suggested that disco/disco-r and btd may have similar roles. The Btd protein has been shown to be required along with the homeodomain-containing protein Empty spiracles (Ems) to specify intercalary identity. Ectopic Ems is capable of transforming regions only where Btd is present, indicating that Btd is necessary for Ems activity. Btd and Ems proteins can interact, and this can occur at the Btd zinc finger domain as well as elsewhere in the protein. It is concluded that Btd and Ems together specify intercalary identity, and that Btd represses phenotypic suppression of Ems. This supports the contention that Ems is an escaped HOM-C gene (Mahaffey, 2001 and references therein).
Though repression of phenotypic suppression may occur, it is proposed that there is a more fundamental role for the proteins encoded by btd and disco/disco-r. It is proposed that these zinc finger-containing proteins are required along with the HOM-C proteins to activate the appropriate target genes necessary to establish segment identity. In the case of disco and disco-r, this is with Dfd and Scr during differentiation of the gnathal lobes. disco and disco-r have a lot in common with the HOM-C genes. They encode spatially restricted transcription factors. Absence of these genes causes a similar phenotype to loss of Dfd and Scr, suggesting a loss of segment identity. It is suggested that, as with the HOM-C genes, disco and disco-r are needed to establish the appropriate transcriptional environment for gnathal segment identity. In an analogous manner, Btd and Ems are required for intercalary identity. Further, since Btd interacts directly with Ems, it seems possible that similar interactions may occur between other HOM-C proteins and zinc finger cofactors. It is tempting to speculate that this occurs with Disco/Disco-r and Dfd and Scr, but this may be a bit premature. Additional studies are necessary to determine if this model is correct, but the similarity of larvae lacking these genes to those lacking Dfd and Scr implies that the disco and disco-r function is crucial for normal pattern formation in the gnathal lobes (Mahaffey, 2001).
The Drosophila disconnected gene is required for the formation of appropriate connections between the larval optic nerve and its target cells in the brain. The disco gene encodes a nuclear protein with two zinc fingers, which suggests that the gene product is a transcription factor. Data supporting this notion. disco expression in the embryonic optic lobe primordium, a group of cells contacted by the developing optic nerve, depends on an autoregulatory feedback loop. Wild-type disco function is required for maintenance of disco mRNA and protein expression in the developing optic lobe. In addition, ubiquitous Disco activity supplied by a heat-inducible gene construct activates expression from the endogenous disco gene specifically in the optic lobe primordium. Consistent with a role of Disco as a transcriptional regulatory protein, portions of the Disco protein are capable of activating the transcription of reporter constructs in a heterologous system. Moreover, the zinc finger portion of Disco binds in vitro to sequences located near the disco transcription unit, suggesting that Disco autoregulates its transcription in the optic lobe primordium by direct binding to a regulatory element in its own promoter (Lee, 1999).
disco is expressed in the embryonic primordia of the adult optic lobes (Lee, 1991). These primordia develop by invagination from each lateral surface of the posterior procephalic lobe. They remain largely nonneuronal until late larval stages, at which time they divide and differentiate to form the adult optic lobes. The timing of disco expression in the optic lobe primordium and the association of the larval optic nerve with these disco-expressing cells suggest that disco acts in the optic lobe primordium to direct proper formation of the larval visual pathway. Moreover, Disco immunoreactivity is specifically absent in the optic lobe region in disco mutant embryos (Lee, 1991). Attempts were made to characterize disco expression in the optic lobe region in greater detail (Lee, 1999).
Wild-type embryos were double-labeled with an anti- Disco polyclonal antiserum (Lee, 1991) and the monoclonal antibody (mAb) 44C11. This monoclonal antibody recognizes Elav, a nuclear antigen specifically expressed in all Drosophila neurons. A confocal image taken from a stage 13 embryo stained with anti-Disco and mAb 44C11 shows that the developing brain hemisphere consists at this stage of many immature neurons that express Elav. The optic lobe primordium, which has invaginated and is in contact with the developing brain, is composed largely of undifferentiated cells, which do not express Elav. A ventral portion of the optic lobe primordium and a group of immature neurons just anterior to the optic lobe primordium express Disco. Double-labeling with anti-Disco and anti-HRP, which, like 44C11, stains developing neurons of the brain hemisphere, also indicates that Disco is expressed in the ventral optic lobe primordium and in adjacent neurons (Lee, 1999).
This study presents evidence that disco feeds back on its own level of expression in the optic lobe primordium. Furthermore, Disco protein has sequence-specific DNA binding activity in vitro and that it contains a glutamine-rich region and a zinc finger domain that can function as a transactivation domain in a yeast assay. These observations, along with the localization of the gene product in the nucleus, implicate Disco in the regulation of gene expression at the level of transcription. Ultimately the importance of disco as a transcription factor in vivo needs to be addressed by the identification of target genes and the respective binding sites, essential for the development of the larval visual system connectivity (Lee, 1999).
Several lines of evidence indicate that the Disco zinc finger domain is a key functional part of the molecule. Two point-mutant alleles of show changes in conserved cysteines in either one of the two zinc fingers (Heilig, 1991). These two alleles have a mutant phenotype that is indistinguishable from the phenotype produced by a deletion of the disco gene. Thus, changes in critical residues in either Disco zinc finger appear to result in a complete loss of gene function. A mutation in one of the two zinc fingers leads to loss of the in vitro DNA binding activity of the Disco protein. Similar mutations in one or the other zinc finger of the yeast ADR1 protein also result in a complete loss of function. Likewise, it has been demonstrated that two of the five zinc fingers found in the PRDI-BF1 protein are necessary and sufficient for proper sequence-specific DNA binding in vitro. Therefore, it may be a general property of this class of zinc finger transcription factors that a minimum of two intact zinc fingers is required for protein function (Lee, 1999).
The zinc finger domain of Disco can function to activate transcription in a heterologous yeast, system and this activity depends on the integrity of the zinc finger sequences. These observations raise the possibility that the zinc fingers present in Disco have a dual role: in DNA binding and in protein-protein interactions required for transcriptional transactivation. Zinc fingers of the C2H2 type found in Disco have been reported to bind both DNA and protein or DNA and RNA. However, the experiments reported here do not address the question of whether the transactivation function of the zinc finger domain detected in yeast is relevant to disco gene function in vivo (Lee, 1999).
A human keratinocyte protein with striking sequence similarity to disco has been described (Tseng, 1992). This protein, Basonuclin, contains six zinc fingers, arranged in three separate pairs. Each of these pairs of zinc fingers is very closely related in sequence to the pair of zinc fingers found in Disco. Outside the zinc finger sequences, Disco and Basonuclin share little sequence similarity. Thus, the evolutionary conservation of the zinc finger sequences indicates that this region plays an important role in the function of the two proteins. It also suggests that the two proteins may have similar activities; for example, they may bind nucleic acids with similar binding specificity or they may regulate a common set of downstream target genes (Lee, 1999).
The maintenance of disco mRNA and protein expression in the optic lobe primordium beyond stage 9 or 10 depends on wild-type disco function. The loss of disco expression in the disco mutant optic lobe is not the result of cell degeneration or mislocalization. Moreover, the loss of disco activity does not appear to result in large-scale changes in cellular identity or differentiation in the developing optic lobe, since the expression of cell type-specific markers is unaffected. Ubiquitously expressed Disco under the control of a heat-inducible promoter activates expression from the endogenous gene specifically in the optic lobe primordium (Lee, 1999).
Taken together, these data suggest a model wherein expression in the optic lobe region is initiated by factors that operate independent of disco activity. Later, an autoregulatory feedback mechanism is required for the maintenance of disco expression in the optic lobe primordia (Lee, 1999).
Maintenance of disco expression in other embryonic tissues, in contrast, is not dependent on disco function. In some tissues in which disco is expressed, genes that control expression have been identified. In leg disc primordia, the homeotic gene Ultrabithorax (Ubx) regulates the expression of Distalless (Dll), which in turn regulates the expression of disco. In larval heart precursor cells, the cardioblasts, disco expression depends on the product of homeodomain-containing gene tinman (Lee, 1999).
Similar autoregulatory mechanisms have been described for a number of transcription factors involved in Drosophila and mammalian development. It is thought that these autoregulatory mechanisms involve transcriptional regulation mediated by direct binding of the protein to discrete elements in the genomic DNA near the corresponding gene. Such direct binding has been best demonstrated for the Drosophila fushi tarazu and Deformed genes (Lee, 1999).
Since disco is likely to encode a transcription factor, it is hypothesized that disco autoregulation might similarly depend on direct binding of Disco to its own promoter region. Consistent with this hypothesis, it was found that Disco binds in vitro to sites in the genomic DNA near the transcription unit. The location of one of these binding sites, roughly 2.5 kb upstream of the disco transcription start, is similar to the location of autoregulatory enhancers defined for the Drosophila Dfd and ftz genes. Further study is needed to determine whether the Disco binding sites identified are necessary and/or sufficient for autoregulation in vivo (Lee, 1999).
disco autoregulation is specifically active in the optic lobe primordium. It is infered that the tissue specificity of disco autoregulation is a consequence of interactions with another factor or factors which are spatially restricted. Such factors may act positively, by promoting disco autoregulation specifically in the optic lobe primordium, or negatively, by inhibiting this activity in other tissues. The activity of another Drosophila zinc finger protein, Glass, is negatively regulated in nonneuronal cells by an unidentified DNA binding protein. At present, the molecular mechanism of the tissue specificity of disco autoregulation is unknown; it may, for example, involve changes in the binding of Disco to DNA target sites or changes in the response of the disco promoter (Lee, 1999).
Although the autoregulatory activity of disco is restricted to the optic lobe primordium, it is likely that disco is active in other tissues. The disco gene is normally expressed in a subset of cells in the peripheral nervous system (PNS) (Lee, 1991) and abnormalities in PNS development are observed in disco mutant embryos (Steller, 1987). In addition, ectopic expression of disco in hs- embryos produces striking defects throughout the nervous system (A. R. Campos and H. Steller, unpublished results). As we show here, however, this ectopic disco activity does not autoactivate disco expression. Therefore, these other activities of the disco gene, which are likely to involve the regulation of the expression of other genes, are not dependent on the same tissue-specific factors as is disco autoregulation (Lee, 1999).
The disco gene product has several features characteristic of transcriptional regulatory proteins and it autoregulates its expression in the optic lobe primordium. What is the function of Disco and what is the role of this autoregulatory mechanism in the development of the larval visual system (Lee, 1999)?
Although the significance of Disco in tissue-specific autoregulation remains unclear, it is noteworthy that this regulatory feedback occurs selectively in the optic lobe primordium, a tissue that appears to play a critical role in larval optic nerve guidance. As an essential intermediate target for the larval optic nerve, the optic lobe primordium must express a set of cell-surface molecules that direct stable interactions with the larval photoreceptor axons. Contact between the optic lobe primordium and the optic nerve is maintained during a period of significant remodeling of the larval head segments. Moreover, during this period, the optic lobe cells separate and invaginate from the head ectoderm, although they remain in an immature state until larval stages when they divide and differentiate to form the adult optic lobes. Thus, the autoregulatory mechanism described in this study may function to maintain the identity of the optic lobe primordium throughout this period of contact with the larval optic nerve. In addition to feeding back on its own expression, Disco may directly regulate the stable expression of certain critical molecules involved in the interaction between optic lobe cells and photoreceptor axons. These putative targets of disco regulatory activity remain to be determined, but they might include genes encoding cell-adhesion or cell-recognition molecules (Lee, 1999).
In addition, disco function is required for the differentiation of a subset of optic lobe cells that are thought to play a essential role in directing optic nerve connectivity. In mutants, the optic lobe pioneers, a group of three neurons that are among the earliest born cells of the optic lobe, are missing or develop abnormally. These neurons are normally located near the point where the larval optic nerve enters the optic lobe primordium and they extend processes which fasciculate with the nerve. These features have led to the suggestion that the OLPs may play a role in guiding the larval optic nerve or promoting the formation of stable contacts between the nerve and the brain. The absence of the OLP cells in disco mutant brains suggests that the aberrant connectivity of the larval optic nerve results from the improper development of the OLPs (Lee, 1999).
The results suggest that novel insights into the mechanisms that direct connectivity of this model neural circuit may be gained by identifying genes regulated by Disco in the optic lobe primordium and in the OLPs. The analysis of one target gene that depends on disco function, namely the gene itself, may provide an initial framework forfuture studies to identify other target genes that encode molecules involved in cell interactions necessary for optic nerve connectivity (Lee, 1999).
Proper development of the larval visual nerve, Bolwig's nerve, of Drosophila requires the wild type function of the disconnected gene. In disco mutants, the nerve does not make stable connections with its targets in the larval brain. The distribution of disco mRNA and protein was examined in embryos and third instar larvae using in situ hybridization and antibody staining respectively. No differences between the distribution patterns of the two products are detected; disco is expressed in many tissues including both neural and non-neural cells. Many of the cells which express disco undergo extensive movement during development as they participate in major morphogenetic movements. Antibody staining shows that the protein is found in the cell nucleus. Products of the disco gene are detected in cells near the terminus of the growing Bolwig's nerve. In embryos homozygous for either of two mutant alleles of disco, the disco protein is absent near the nerve terminus, although protein distribution elsewhere is indistinguishable from wild type (Lee, 1991)
Mutations in the disco gene prevent the establishment of stable connections between the larval optic nerves, the Bolwig's nerves, and their target cells in the brain during embryonic development. The failure of this initial connection is associated with aberrant development of the optic lobes which are largely degenerate in the mutant adult fly. In order to understand the role of disco in establishing this connection, the disco gene was isolated and characterized. A 22 kb DNA fragment can completely rescue the mutant phenotype. A single transcript, 2.9 kb in length, is found in this region and is expressed throughout development of the fly. The nucleotide sequence of the disco gene was found to be unique when compared with sequences in a number of databases. The predicted amino acid sequence contains a region with similarity to the consensus established for the zinc finger motif. Mobilization of a P-element inserted near the gene resulted in the deletion of the 5' end of the gene and produced flies indistinguishable from those carrying the disco allele (Heilig, 2001).
Search PubMed for articles about Drosophila Disconnected
Bucher, G., and Klingler, M. (2004) Divergent segmentation mechanism in the short germ insect Tribolium revealed by giant expression and function. Development 131: 1729-1740. PubMed ID: 15084458
Harding, K., and Levine, M. (1988). Gap genes define the limits of antennapedia and bithorax gene expression during early development in Drosophila. EMBO J. 7: 205-214. PubMed ID: 2896123
Heilig, J. S., et al. (1991). Isolation and characterization of the disconnected gene of Drosophila melanogaster. EMBO J. 10(4): 809-815. PubMed ID: 1901262
Larsen, C. W., et al. (2003). Segment boundary formation in Drosophila embryos. Development 130: 5625-5635. PubMed ID: 14522878
Lee, K. J. et al. (1991). Expression of the disconnected gene during development of Drosophila melanogaster. EMBO J. 10: 817-826. PubMed ID: 1901263
Lee, K. J., et al. (1999). Autoregulation of the Drosophila disconnected gene in the developing visual system. Dev. Biol. 214: 385-398. PubMed ID: 10525342
Liu, P. Z., and Kaufman, T. C. (2004). hunchback is required for suppression of abdominal identity, and for proper germband growth and segmentation in the intermediate germband insect Oncopeltus fasciatus. Development 131: 1515-1527. PubMed ID: 14998925
Mahaffey, J. W., Griswold, C. M. and Cao, Q. M. (2001). The Drosophila genes disconnected and -related are redundant with respect to larval head development and accumulation of mRNAs from deformed target genes. Genetics 157: 225-236. PubMed ID: 11139504
Mohler, J., Eldon, E. D. and Pirrotta, V. (1989). A novel spatial transcription pattern associated with the segmentation gene, giant, of Drosophila. EMBO J. 8: 1539-1548. PubMed ID: 2504582
Patel, M., et al. (2007). The appendage role of insect genes and possible implications on the evolution of the maggot larval form. Dev. Biol. 309: 56-69. PubMed ID: 17643406
Petschek, J. P. and Mahowald, A. P. (1990). Different requirements for l(1) giant in two embryonic domains of Drosophila melanogaster. Dev. Genet. 11: 88-96. PubMed ID: 2113845
Robertson, L. K., Bowling, D. B., Mahaffey, J. P., Imiolczyk, B. and Mahaffey, J. W. (2004). An interactive network of zinc-finger proteins contributes to regionalization of the Drosophila embryo and establishes the domains of HOM-C protein function. Development 131: 2781-2789. PubMed ID: 15142974
Sanders, L. R., Patel, M. and Mahaffey, J. W. (2008). The Drosophila gap gene giant has an anterior segment identity function mediated through disconnected and teashirt. Genetics 179(1): 441-53. PubMed ID: 18493063
Steller, H. et al. (1987). Disconnected: a locus required for neuronal pathway formation in the visual system of Drosophila. Cell 50: 1139-1153. PubMed ID: 3113740
Tseng, H. and Green, H. (1992). Basonuclin: a keratinocyte protein with multiple paired zinc fingers. Proc. Natl. Acad. Sci. 89(21): 10311-5. PubMed ID: 1332044
date revised: 30 November 2009
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